Biopolymers, such as nucleic acids, proteins and polysaccharides play important roles in fundamental aspects of cellular functions. These biomolecules are capable of storing as well as transmitting biological information that involves intra- and intercellular events. Unlike polysaccharides, the biological roles and functions of nucleic acids and proteins are relatively well understood and are better appreciated by the scientific community. While nucleic acid and polypeptide-derived natural biopolymers are linear in nature and chemically defined, polysaccharides are structurally more complex. This structural and stereochemical diversity results in a rich content of information in relatively small molecules.
In recent years, efforts are being made towards understanding the biological roles of glycoconjugates (commonly known as glycobiology) in the modulation of protein function, fertilization, chronic inflammation, immune responses and cancer metastasis. It is now well accepted that glycoconjugates present on host cell surfaces provide specific binding sites for the attachment of bacterial and viral pathogens leading to infectious diseases. In addition, it has been demonstrated that the oligosaccharide moieties of several complex glycoconjugates present on tumor cell surfaces have unique structural features. These moieties are attractive targets for developing chemically well-defined, synthetic vaccines for cancer and the design of specific delivery of anticancer drugs on tumor cell surfaces.
The growing appreciation of the key roles of oligosaccharides and glycoconjugates in fundamental life sustaining processes has stimulated a need for access to usable quantities of these materials. Glycoconjugates are difficult to isolate in homogeneous form from living cells since they exist as microheterogeneous mixtures. Further, the purification of these compounds, when possible, is at best tedious and generally provides only very small amounts of the compounds. The difficulties associated with isolation of oligo- and poly-saccharides and glycoconjugates from natural sources present a major opportunity for the development and exploitation of chemical synthesis.
The invention of solid phase peptide synthesis dramatically influenced the strategy for the synthesis of these biopolymers. The preparation of structurally defined oligopeptides has benefited greatly from the feasibility of conducting their assembly on various polymer supports. The advantages of solid matrix-based synthesis, in terms of allowing for an excess of reagents to be used and in the facilitation of purification are now well appreciated. However, the level of complexity associated with the synthesis of an oligosaccharide on a polymer support dwarfs that associated with the other two classes of repeating biooligomers. First, the need to differentiate similar functional groups (hydroxyl or amino) in oligosaccharide construction is much greater than the corresponding needs in the synthesis of oligopeptides or oligonucleotides. Furthermore, in these latter two cases, there is no stereoselection associated with construction of the repeating amide or phosphate bonds. In contrast, each glycosidic bond to be fashioned in a growing oligosaccharide ensemble constitutes a new locus of stereogenicity.
The development of protocols for the solid support synthesis of oligosaccharides and glycopeptides requires solutions to several problems. First, the nature of the support material is relevant. The availability of methods for attachment of the carbohydrate from either its “reducing” or “non-reducing” end would be advantageous. Also, selection of a linker which is stable during the synthesis, but can be cleaved easily when appropriate, is critical. A protecting group strategy that allows for high flexibility is desirable. Also important is the matter of stereospecific and high yielding coupling reactions.
Besides synthesis, another major obstacle in the field of glycobiology is monitoring of the synthesis of oligosaccharides and glycoconjugates. Analytical tools for on-bead characterization include High-Resolution Magic Angle Spinning NMR spectroscopy (HRMAS), FT-IR, and Gated-Decoupling 13C NMR Spectroscopy. While these monitoring methods have been successful, they are not amenable to automated synthesis. Other current methods of monitoring oligosaccharide synthesis include solid state NMR, bead IR, and UV monitoring of cleavage products.
There are currently only five labs in the world that can synthesize complex oligosaccharides. Only one of these labs has successfully automated the process by adapting a conventional peptide synthesizer. To date, none have attempted to design molecules that allow colorimetric monitoring of the process.
In the past, oligonucleotide synthesis in solution has been carried out mainly by the conventional phosphotriester approach that was developed in the 1970s (Reese, C. B., Tetrahedron 1978, 34, 3143-3179; Kaplan, B. E.; Itakura, K. in Synthesis and Applications of DNA and RNA, Narang, S. A., Ed., Academic Press, Orlando, 1987, pp. 9-45). This approach can also be used in solid phase synthesis but coupling reactions are somewhat faster and coupling yields are somewhat greater when phosphoramidite monomers are used. This is why automated solid phase synthesis has been based largely on the use of phosphoramidite building blocks; it is perhaps also why workers requiring relatively large quantities of synthetic oligonucleotides have decided to attempt the scaling-up of phosphoramidite-based solid phase synthesis.
Three main methods, namely the phosphotriester (Reese, Tetrahedron, 1978), phosphoramidite (Beaucage, S. L. in Methods in Molecular Biology, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 33-61) and H-phosphonate (Froehler, B. C. in Methods in Molecular Biology, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 63-80; see also WO94/15946 and Dreef, C. E. in Rec. Tray. Chim. Pays-Bas, 1987, 106, p 512) approaches have proved to be effective for the chemical synthesis of oligonucleotides. While the phosphotriester approach has been used most widely for synthesis in solution, the phosphoramidite and H-phosphonate approaches have been used almost exclusively in solid phase synthesis.
Perhaps the most widely used strategy for the synthesis of oligodeoxyribonucleotides in solution involves a coupling reaction between a protected nucleoside or oligonucleotide 3′-(2-chlorophenyl)phosphate (Chattopadhyaya, J. B.; Reese, C. B. Nucleic Acids Res., 1980, 8, 2039-2054) and a protected nucleoside or oligonucleotide with a free 5′-hydroxy function to give a phosphotriester. A coupling agent such as 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-1H-triazole (MSNT) (Reese, C. B.; Titmas, R. C.; Yau, L. Tetrahedron Lett., 1978, 2727-2730) is required. This strategy has also been used in the synthesis of phosphorothioate analogues. Coupling is then effected in the same way between a protected nucleoside or oligonucleotide 3′-S-(2-cyanoethyl or, for example, 4-nitrobenzyl)phosphorothioate (Liu, X.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1, 1995, 1685-1695) and a protected nucleoside or oligonucleotide with a free 5′-hydroxy function.
There is a need in the art for a fast and simple method of colorimetrically monitoring reactions on a solid support for automated chemical synthesis that may be used in addition to other known techniques for monitoring such synthesis.
It is a primary objective of the present invention to provide a novel protecting group to provide colorimetric monitoring for solid and solution phase synthesis.
It is a further objective of the present invention to provide 4-(ortho-nitrophthalimido)butyric acid (NPB) to allow colorimetric monitoring for solid and solution phase synthesis.
It is a further objective of the present invention to provide p-nitrophenyl carbonate (NPC) to allow colorimetric monitoring for solid and solution phase synthesis.
It is a further objective of the present invention to provide a method for assaying to determine the amount and type of a particular chemical produced during solid and solution phase synthesis.
It is yet a further objective of the present invention to provide a method for determining and confirming the presence or amount of a particular chemical produced during solid and solution phase synthesis.
It is another objective of the present invention to provide a kit for determining the presence or amount of a particular chemical produced during solid and solution phase synthesis.
It is still a further objective of the present invention to provide a method of monitoring reactions on solid support for automated synthesis.
It is a further objective of the present invention to provide a method of colorimetric monitoring reactions on solid support for automated synthesis that is simpler and faster than previous techniques.
The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereafter.